This project studies the regulation of expression of genes encoding lens fiber membrane channel proteins. We are presently focusing on the regulation of expression of the gene encoding MIP, the major intrinsic protein of the lens fiber membrane, that is specifically expressed in the ocular lens fibers and belongs to an ancient superfamily of transmembrane channel proteins. We are studying the cis regulatory elements responsible for the lens specificity and developmental regulation of the MIP gene. We are fusing 5' flanking sequences of the human MIP gene to the reporter chloramphenicol acetyltransferase (CAT) gene and analyzing CAT gene expression in transient assays in primary cultures of lens cells. Deletion analysis of the 5' flanking sequence of the human MIP gene suggests that negative regulatory elements are located within the sequence -2840/-254. The human MIP sequence -70/-40 appears to contain an important regulatory element for the activity of the MIP promoter. This sequence contains one of the domains that interacts with Sp1 and AP2 transcription factors by DNase I footprinting analysis; it also forms a complex with chicken lens nuclear extracts that appear as a retarded band in mobility shift assays. Mutation at positions -49/-51 affect binding of this nuclear factor in vitro, and deletion of the sequence -70/-49 abolishes promoter activity in transfected lens cells. These studies will further our understanding of the regulation of the MIP gene expression in the lens.